Combination of cross-linked hyaluronic acids and method of preparing the same

ABSTRACT

Provided are a method of preparing a cross-linked hyaluronic acid having an elasticity of about 50 to about 200 Pa and a viscosity of about 20 to about 100 Pa, including cross-linking hyaluronic acid with an epoxy-based cross-linking agent having at least two epoxy functional groups in an ethanol-containing aqueous alkaline; a method of preparing a cross-linked hyaluronic acid having an elasticity of about 400 to about 800 Pa and a viscosity of about 40 to about 100 Pa, including cross-linking the same cross-linked hyaluronic acid with the same epoxy-based cross-linking agent in an aqueous alkaline solution; and a combination of the cross-linked hyaluronic acids one having an elasticity of about 50 to about 200 Pa and a viscosity of about 20 to about 100 Pa and the other having an elasticity of about 400 to about 800 Pa and a viscosity of about 40 to about 100 Pa.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a National Phase Application filed under 35 U.S.C.371 as a national stage of PCT/KR2016/005819, filed Jun. 1, 2016, andclaims the benefit of Korean Patent Application No. 10-2016-0011843,filed on Jan. 29, 2016, and Korean Patent Application No.10-2016-0012519, filed on Feb. 1, 2016, in the Korean IntellectualProperty Office, the disclosures of which are incorporated herein intheir entireties by reference.

TECHNICAL FIELD

The present disclosure relates to a preparation method of a cross-linkedhyaluronic acid and a combination of cross-linked hyaluronic acidshaving a viscoelasticity appropriate for use in the living body,comprising the cross-linked hyaluronic acid prepared using the samemethod.

BACKGROUND ART

Hyaluronic acids are a biopolymer material including linearly linkedrepeating units consisting of N-acetyl-D-glucosamine and D-glucuronicacid, and are also known to be prevalent in animal placenta, vitreoushumour, synovial fluid, rooster combs, and the like. Hyaluronic acidsare also known to be produced via fermentation by microorganisms of theStreptococcus spp. (for example, Streptococcus equi, or Streptococcuszooepidemicus) or the Staphylococcus spp.

Synvisc-One®, a cross-linked hyaluronic acid injection, which provideits effect lasting up to 6 months with one injection, are commerciallyavailable in the U.S. Synvisc-One® includes a cross-linked hyaluronicacid obtained by extracting hyaluronic acids from rooster combs with aformalin-containing aqueous solution, having a low viscoelasticity dueto the light cross-linking of proteins connected to hyaluronic acidswith formalin (U.S. Pat. No. 4,713,448). The lightly cross-linkedhyaluronic acid is combined with its further cross-linked hyaluronicacid having increased viscoelasticity prepared by further cross-linkingthe lightly cross-linked hyaluronic acid using divinyl sulfone (DVS) asa cross-linking agent, thereby preparing a combination of cross-linkedhyaluronic acids (Synvisc-One®) having appropriate viscoelasticity forapplying to the joint cavity in the human body.

However, hyaluronic acids of animal origin such as from rooster combsmay have a quality control problem due to animal-origin viruses andinconsistent quality of source material.

DETAILED DESCRIPTION OF THE INVENTION Technical Problem

The present disclosure provides a method of preparing a cross-linkedhyaluronic acid having a low viscoelasticity.

The present disclosure provides a combination of cross-linked hyaluronicacids having an appropriate viscoelasticity applicable to the humanbody.

The present disclosure provides a biocompatible material including thecombination of cross-linked hyaluronic acids.

The present disclosure provides a method of preparing the combination ofcross-linked hyaluronic acids having an appropriate viscoelasticityapplicable to the living body.

Technical Solution

According to an aspect of the present disclosure, there is provided amethod of preparing a cross-linked hyaluronic acid having an elasticityof about 50 Pa to about 200 Pa and a viscosity of about 20 Pa to about100 Pa, the method including cross-linking hyaluronic acid with anepoxy-based cross-linking agent having at least two epoxy functionalgroups in an ethanol-containing aqueous alkaline solution.

According to another aspect of the present disclosure, there is provideda method of preparing a cross-linked hyaluronic acid having anelasticity of about 400 Pa to about 800 Pa and a viscosity of about 40Pa to about 100 Pa, the method including further cross-linking thecross-linked hyaluronic acid prepared using the above-described methodwith an epoxy-based cross-linking agent having at least two epoxyfunctional groups in an aqueous alkaline solution.

According to another aspect of the present disclosure, there is provideda combination of cross-linked hyaluronic acids, the combinationincluding a cross-linked hyaluronic acid having an elasticity of about50 to about 200 Pa and a viscosity of about 20 to about 100 Pa, and across-linked hyaluronic acid having an elasticity of about 400 to about800 Pa and a viscosity of about 40 to about 100 Pa.

According to another aspect of the present disclosure, there is provideda combination of cross-linked hyaluronic acids, the combinationincluding: a cross-linked hyaluronic acid having an elasticity of about50 to about 200 Pa and a viscosity of about 20 to about 100 Pa preparedusing a method including cross-linking hyaluronic acid with anepoxy-based cross-linking agent having at least two epoxy functionalgroups in an ethanol-containing aqueous alkaline solution; and

a cross-linked hyaluronic acid having an elasticity of about 400 toabout 800 Pa and a viscosity of about 40 to about 100 Pa prepared usinga method including cross-linking the cross-linked hyaluronic acid havingan elasticity of about 50 to about 200 Pa and a viscosity of about 20 toabout 100 Pa with an epoxy-based cross-linking agent having at least twoepoxy functional groups in an aqueous alkaline solution.

According to another aspect of the present disclosure, there is provideda biocompatible material including any of the above-describedcombinations of cross-linked hyaluronic acids.

According to another aspect of the present disclosure, there is provideda method of preparing a combination of cross-linked hyaluronic acids,the method including combining the cross-linked hyaluronic acid havingan elasticity of about 50 to about 200 Pa and a viscosity of about 20 toabout 100 Pa with the cross-linked hyaluronic acid having an elasticityof about 400 to about 800 Pa and a viscosity of about 40 to about 100Pa, wherein the combining ratio is adjusted to meet a targetviscoelasticity of the combination of cross-linked hyaluronic acids tobe prepared.

Advantageous Effects

According to the one or more embodiments of the present disclosure, in amethod of preparing a cross-linked hyaluronic acid using an epoxy-basedcross-linking agent in an aqueous alkaline solution, ethanol is added tothe aqueous alkaline solution, so that the prepared cross-linkedhyaluronic acid may have a low viscoelasticity. The viscoelasticity ofthe cross-linked hyaluronic acid may be adjusted depending on thereaction conditions. A combination of cross-linked hyaluronic acidshaving a desired viscoelasticity may be prepared by combining thecross-linked hyaluronic acid having a low viscoelasticity with thecross-linked hyaluronic acid having a high viscoelasticity obtainedthrough further cross-linking of the low-viscoelasticity cross-linkedhyaluronic acid. Therefore, a biocompatible combination of cross-linkedhyaluronic acids having a viscoelasticity appropriate for use in thehuman body, and in particular, having a similar viscoelasticity similarto the human synovial fluid may be prepared for effective use inosteoarthritis treatment.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flowchart illustrating a method of preparing a combinationof cross-linked hyaluronic acids, according to an embodiment of thepresent disclosure.

FIG. 2 is a graph of particle size distribution as a result of measuringparticle sizes of a combination of cross-linked hyaluronic acidsprepared with the primary cross-linked product and secondarycross-linked product of high-molecular weight (about 2.5 MDa) hyaluronicacids in a mixed ratio of about 9:1 by weight, according to anembodiment of the present disclosure.

FIG. 3 is a graph of cell survival ratio illustrating the results of theMTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium) assay on thecombination of cross-linked hyaluronic acids prepared with the primarycross-linked product and secondary cross-linked product ofhigh-molecular weight hyaluronic acids in a mixed ratio of about 9:1 byweight, according to an embodiment of the present disclosure, andpositive (Teflon) and negative (Latex) control groups.

MODE FOR INVENTION

Unless otherwise defined, all terms (including technical and scientificterms) used herein have the same meaning as commonly understood by oneof ordinary skill in the art to which this invention belongs. Althoughexemplary methods or materials are listed herein, other similar orequivalent ones are also within the scope of the present invention. Anynumerical expression herein may be construed as the meaning ofapproximation, such as “about”, unless otherwise defined. Allpublications disclosed as references herein are incorporated in theirentirety by reference.

The inventors of the present disclosure attempted to prepare lightlycross-linked hyaluronic acids from purified source material ofhyaluronic acids originating from microorganisms, but failed due to lowprotein content of the purified hyaluronic acids of microorganismorigin. As a result of research into a method of light cross-linking ofpurified hyaluronic acids of microorganism origin having low proteincontent, the inventors found that lightly cross-linked hyaluronic acidshaving a novel range of low viscoelasticity are obtained by introducingethanol to an aqueous alkaline solution where a conventional hyaluronicacid cross-linking reaction with a multifunctional epoxy-basedcross-linking agent takes place. The inventors also found thatcross-linked hyaluronic acids having an increased viscoelasticity may beobtained by a secondary cross-linking reaction of the lightlycross-linked hyaluronic acids having a low viscoelasticity with amultifunctional epoxy-based cross-linking agent.

An aspect of the present disclosure provides a cross-linked hyaluronicacid having an elasticity of 50 to 200 Pa and a viscosity of 20 to 100Pa.

The cross-linked hyaluronic acids are not limited only to thosecross-linked hyaluronic acids obtained by a specific cross-linkingmethod.

In some embodiments, the cross-linked hyaluronic acid may be prepared bya method including a step of cross-linking hyaluronic acids with anepoxy-based cross-linking agent having at least two epoxy functionalgroups in an ethanol-containing aqueous alkaline solution. This will bedescribed later in more detail.

The cross-linked hyaluronic acid having an elasticity of 50 to 200 Paand a viscosity of 20 to 100 Pa may be used as any biocompatiblematerial using hyaluronic acids, for example, selected from the groupconsisting of arthritis treatment implants, wrinkle fillers, cosmeticfillers, and drug carriers. The cross-linked hyaluronic acid may be usedas a more appropriate biocompatible material in a combination with across-linked hyaluronic acid having an increased viscoelasticity thatmay be prepared via a further cross-linking reaction, if required.

Another aspect of the present disclosure provides a method of preparinga cross-linked hyaluronic acid having an elasticity of 50 to 200 Pa anda viscosity of 20 to 100 Pa, the method including cross-linkinghyaluronic acids with an epoxy-based cross-linking agent having at leasttwo epoxy functional groups in an ethanol-containing aqueous alkaline.

Another aspect of the present disclosure provides a method of preparinga cross-linked hyaluronic acid having an elasticity of 400 to 800 Pa anda viscosity of 40 to 100 Pa, the method including further cross-linkingthe cross-linked hyaluronic acid prepared using the above-describedmethod with an epoxy-based cross-linking agent having at least two epoxyfunctional groups in an aqueous alkaline solution.

As used herein, the cross-linked hyaluronic acid having an elasticity of50 to 200 Pa and a viscosity of 20 to 100 Pa, prepared using theabove-describe method, is also referred to as a “primary cross-linkedproduct”, and the cross-linked hyaluronic acid having an elasticity of400 to 800 Pa and a viscosity of 40 to 100 Pa, prepared by furthercross-linking the primary cross-linked product, is also referred to as a“secondary cross-linked product.” The primary cross-linked product maybe in the form of hyaluronic acid gel, and the secondary cross-linkedproduct may be in the form of hyaluronic acid particles.

As used herein, the term “hyaluronic acid” may be construed as any ofhyaluronic acid, a salt of hyaluronic acid, or any mixtures thereof. Thesalt of hyaluronic acid may be any biocompatible salt form, for example,selected from the group consisting of sodium hyaluronate, potassiumhyaluronate, magnesium hyaluronate, zinc hyaluronate, cobalthyaluronate, tetrabutylammonium hyaluronate, and any combinationsthereof. In some embodiments, the salt of hyaluronic acid may be sodiumhyaluronate.

The hyaluronic acid may have a molecular weight of about 100,000 toabout 6,000,000. A viscoelasticity of the cross-linked hyaluronic acidmay vary depending on the molecular weight of the hyaluronic acid.

In some embodiments, the hyaluronic acid may be sodium hyaluronatehaving a molecular weight of about 100,000 to about 6,000,000.

The hyaluronic acid may include any hyaluronic acids known in the art towhich the present disclosure pertains. The hyaluronic acid may beobtained from any sources. The hyaluronic acid may be obtained, forexamples, from animal sources (for example, animal placenta, roostercombs, or the like) or any microorganisms that may produce hyaluronicacids through fermentation (for example, microorganisms of theStaphylococcus spp., the Streptococcus spp., or the like).

In some embodiments, the hyaluronic acid may be hyaluronic acid ofmicroorganism origin, for example, of the Staphylococcus spp.microorganism origin. Hyaluronic acids of microorganism origin may befree of the problems with animal-origin hyaluronic acids, such as virusor inconsistent quality of source material, and thus may be advantageousin view of quality control in drug preparation.

The epoxy-based cross-linking agent having at least two epoxy functionalgroups may be any epoxy-based cross-linking agent having at least twoepoxy functional groups known in the art, for example, selected from thegroup consisting of 1,4-butanediol diglycidyl ether (BDDE), ethyleneglycol diglycidyl ether (EGDGE), 1,6-hexanediol diglycidyl ether,propylene glycol diglycidyl ether, polypropylene glycol diglycidylether, polytetramethylene glycol diglycidyl ether, neopentyl glycoldiglycidyl ether, polyglycerol polyglycidyl ether, digylcerolpolyglycidyl ether, glycerol polyglycidyl ether, trimethylpropanepolyglycidyl ether, 1,2-(bis(2,3-epoxypropoxy)ethylene, pentaerythritolpolyglycidyl ether, sorbitol polyglycidyl ether, and any combinationsthereof.

In some embodiments, the epoxy-based cross-linking agent having at leasttwo epoxy functional groups may be BDDE, EGDGE, 1,6-hexanedioldiglycidyl ether, or any combinations thereof.

In some embodiments, the amount of the epoxy-based cross-linking agentthat may be reacted with the hyaluronic acid or cross-linked hyaluronicacid may be in a range of about 10 μl/g to about 100 μl/g, and in someembodiments, about 20 μl/g to about 100 μl/g with respect to thehyaluronic acid or cross-linked hyaluronic acid. When the amount of theepoxy-based cross-linking agent that reacted with the hyaluronic acid orcross-linked hyaluronic acid is less than these ranges, gel formationmay not occur in preparation of the primary cross-linked product orcross-linked particles may not be obtained in preparation of thesecondary cross-linked product. When the amount of the epoxy-basedcross-linking agent that reacted with the hyaluronic acid orcross-linked hyaluronic acid is more than these ranges, a primarycross-linked product may be obtained in the form of particles, not anintended gel having a low viscoelasticity.

The ethanol-containing aqueous alkaline solution may be an aqueousalkaline solution containing about 5 to 13% w/w of ethanol. Theviscosity of the cross-linked hyaluronic acid may be controlled with theconcentration of the ethanol. When the concentration of the ethanol iswithin this range, sodium hyaluronate may be stably mixed with theethanol, not extracted by the ethanol, so that a cross-linking reactionmay take place.

The aqueous alkaline solution may be an aqueous alkaline solution ofabout pH 9 to 13. The aqueous alkaline solution may be any aqueousalkaline solution known to be available in preparation of cross-linkedhyaluronic acids. For example, the aqueous alkaline solution may be anaqueous sodium hydroxide solution, an aqueous potassium hydroxidesolution, or ammonia water. For example, the aqueous alkaline solutionmay be an aqueous sodium hydroxide solution.

In some embodiments, the ethanol-containing aqueous alkaline solutionmay be an about 0.7 to about 1.3% w/w aqueous sodium hydroxide solutioncontaining about 5 to about 13% w/w of ethanol.

As a result of the experiment, hyaluronic acid gel was formed only whenhyaluronic acids reacted with an epoxy-based cross-linking agent havingat least two epoxy functional groups in an aqueous alkaline solutioncontaining ethanol, but not in an aqueous alkaline solution containingother alcohols, for example, methanol or isopropanol. It was also foundthat the viscoelasticity of the formed hyaluronic acid gel variesdepending on the reaction time. In other words, it was found thathyaluronic acid hydrogel may be formed when the aqueous alkalinesolution contains especially ethanol among organic solvents. It was alsofound that the reaction time may be appropriately varied to formhyaluronic acid gel having a desired viscoelasticity.

The cross-linking reaction may be performed in the presence of ethanolat a temperature range higher than room temperature for rapidcross-linking reaction. This rapid cross-linking reaction may inducelight cross-linking. The temperature range higher than room temperaturemay be from about 40 to about 60

.

The primary cross-linked product may be a cross-linked hyaluronic acidin hydrogel form. The secondary cross-linked product obtained throughfurther cross-linking of the primary cross-linked product may be across-linked hyaluronic acid in particle form.

According to an experimental result, it was found that a combination ofcross-linked hyaluronic acids having a desired viscoelasticity may beobtained by combination of the primary cross-linked product and thesecondary cross-linked product.

Another aspect of the present disclosure provides a combination ofcross-linked hyaluronic acids including a low-viscoelasticitycross-linked hyaluronic acid having an elasticity of 50 to 200 Pa and aviscosity of 20 to about 100 Pa, and a high-viscoelasticity cross-linkedhyaluronic acids having an elasticity of 400 to 800 Pa and a viscosityof 40 to 100 Pa.

The low-viscoelasticity cross-linked hyaluronic acid and thehigh-viscoelasticity cross-linked hyaluronic acid are not limited tocross-linked hyaluronic acids prepared using a specific cross-linkingmethod. In some embodiments, the low-viscoelasticity cross-linkedhyaluronic acid and the high-viscoelasticity cross-linked hyaluronicacid may be the primary cross-linked product of hyaluronic acids and thesecondary cross-linked product of hyaluronic acids, respectively,prepared using the above-described methods of preparing cross-linkedhyaluronic acids.

A ratio of combination of the low-viscoelasticity cross-linkedhyaluronic acid and the high-viscoelasticity cross-linked hyaluronicacid may be adjusted depending on a desired viscoelasticity of thecombination of cross-linked hyaluronic acids.

Another aspect of the present disclosure provides a combination ofcross-linked hyaluronic acids including a primary cross-linked productof hyaluronic acids having an elasticity of about 50 to about 200 Pa anda viscosity of about 20 to about 100 Pa, and a secondary cross-linkedproduct of hyaluronic acids having an elasticity of about 400 to about800 Pa and a viscosity of about 40 to about 100 Pa.

A ratio of combination of the primary cross-linked product and thesecondary cross-linked product may be adjusted depending on a desiredviscoelasticity of the combination of cross-linked hyaluronic acids.

Another aspect of the present disclosure provides a biocompatiblematerial including a combination of cross-linked hyaluronic acidsaccording to any of the above-described embodiments.

The biocompatible material may be a biocompatible material usinghyaluronic acids, for example, selected from the group consisting ofarthritis treatment implants, wrinkle fillers, cosmetic fillers, anddrug carriers. The biocompatible material may have a targetviscoelasticity that may vary depending on its use. A ratio ofcombination of the low-viscoelasticity cross-linked hyaluronic acid andthe high-viscoelasticity cross-linked hyaluronic acid in the combinationof cross-linked hyaluronic acids, or a ratio of combination of theprimary cross-linked product of hyaluronic acids and the secondarycross-linked product of hyaluronic acids in the combination ofcross-linked hyaluronic acids may be adjusted depending on a targetviscoelasticity of the combination of cross-linked hyaluronic acids.

In some embodiments, the combination of cross-linked hyaluronic acidsmay be a combination of cross-linked hyaluronic acids having anelasticity of about 100 to about 150 Pa and a viscosity of about 20 toabout 60 Pa, which may result in a viscoelasticity appropriate for useas a synovial fluid supplement in the human body. It is known that aviscoelasticity similar to that of human synovial fluid may be obtainedat an elasticity of about 100 to about 150 Pa and a viscosity of about20 to about 60 Pa (Balazs, E. A., “The physical properties of synovialfluid and the special role of hyaluronic acid,” Disorders of the Knee 2(1974): 61-74). To obtain a combination of cross-linked hyaluronic acidshaving such a level of viscoelasticity, the ratio of combination of thelow-viscoelasticity cross-linked hyaluronic acid and thehigh-viscoelasticity cross-linked hyaluronic acid, or the ratio ofcombination of the primary cross-linked product and the secondarycross-linked product may be varied according to the molecular weight ofthe hyaluronic acids.

In an experiment, a high-molecular weight (about 2.5 MDa) hyaluronicacid and a low-molecular weight (about 0.9 MDa) hyaluronic acid wereeach subjected to a cross-linking reaction to obtain primarycross-linked products. The resulting primary cross-linked products wereeach subjected to a further cross-linking reaction to obtain a secondarycross-linked product. The primary cross-linked product and the secondarycross-linked product were combined in different ratios to preparecombinations of cross-linked hyaluronic acids, followed by aviscoelasticity measurement. As a result, the primary cross-linkedproducts were found to have a different viscoelasticity depending onwhether a high-molecular weight hyaluronic acid or a low-molecularweight hyaluronic acid was used as a start material. Furthermore, thesecondary cross-linked products were found to have a significantlyincreased viscoelasticity, compared to that of their primarycross-linked product, irrespective of the molecular weight of the startmaterial. The combination of cross-linked hyaluronic acids also each hada different viscoelasticity depending on the ratio of combination of theprimary cross-linked product and the secondary cross-linked product(refer to Experimental Example 2).

To obtain a combination of cross-linked hyaluronic acids having adesired viscoelasticity, the ratio of combination of the primarycross-linked product and the secondary cross-linked product may varydepending on the molecular weight of hyaluronic acid source used as thestart material. A combination ratio of about 9:1 by weight of theprimary cross-linked product and the secondary cross-linked productthereof for the case of a high-molecular weight (about 2.5 MDa)hyaluronic acid source being used as the start material, or acombination ratio of 8:2 by weight of the primary cross-linked productand the secondary cross-linked product thereof for the case of alow-molecular weight (about 0.9 MDa) hyaluronic acid source being usedas the start material may obtain a combination of cross-linkedhyaluronic acids having a viscoelasticity range (at an elasticity ofabout 100 to about 150 Pa and a viscosity of about 20 to 60 Pa)appropriate for use as arthritis treatment implants (refer toExperimental Example 2).

In some embodiments, the combination of cross-linked hyaluronic acidsmay include the primary cross-linked product of hyaluronic acids havingan elasticity of 50 to 200 Pa and a viscosity of 20 to 100 Pa and thesecondary cross-linked product of hyaluronic acids having an elasticityof 400 to 800 Pa and a viscosity of 40 to 100 Pa in a weight ratio ofabout 8:2 to about 9:1.

In some embodiments, the combination of cross-linked hyaluronic acidsmay be in the form of particles, obtained by particlization, having anaverage particle size of about 500 to about 750 μm. The particlizationmay be performed by a general pulverization process after combination ofthe primary cross-linked product and the secondary cross-linked product.

FIG. 1 is a flowchart illustrating a method of preparing a combinationof cross-linked hyaluronic acids, according to an embodiment of thepresent disclosure. This preparation method now will be described below.

Referring to FIG. 1, step (a) is preparing a primary cross-linkedproduct of hyaluronic acids in hydrogel form. For example, sodiumhyaluronate (also abbreviated to “HA”) used in step (a) may be a productprepared by fermentation with a Streptococcus spp. microorganism(available from Hanmi Pharm Co., Ltd.). In particular, step (a1) ismixing a sodium hyaluronate-containing buffer with an ethanol-containingaqueous sodium hydroxide solution. A viscoelasticity of a resultinghyaluronic acid hydrogel may vary depending on a mixing ratio of ethanoland sodium hydroxide in step (a1). In the presence of about 5 to about13% w/w of ethanol in an about 0.7 to about 1.3% w/w aqueous sodiumhydroxide solution, sodium hyaluronate may be stably mixed with theethanol, not extracted thereby, so that a cross-linking reaction maytake place. Step (a2) is mixing sodium hyaluronate with a cross-linkingagent. The amount of the cross-linking agent may be about 10 μl to about100 μl per unit gram of sodium hyaluronate. When the amount of thecross-linking agent is less than this range, a cross-linked hyaluronicacid having a desired viscoelasticity may not be obtained. Step (a3) isa process of cross-linking reaction of sodium hyaluronate with across-linking agent in the ethanol-containing aqueous alkaline solution.In this step, rapid cross-linking reaction may be performed due to areaction temperature higher than room temperature and the presence ofethanol in a reactant, so that a light cross-linking may be induced.This reaction step may be performed in an about 40 to about 60

oven for about 4 to about 6 hours. The resulting cross-linked hyaluronicacids in hydrogel form may be subjected to dialysis in a buffer (step(a4)), followed by a neutralization reaction with distilled water (step(a5)) and then precipitation of hyaluronic acid as hydrogel may beinduced with about 95% ethanol (step (a6)) thereby obtaining across-linked hyaluronic acid in powder form.

Step (b) is preparing a secondary cross-linked product of hyaluronicacids in the form of particles through further cross-linking of theprimary cross-linked product of hyaluronic acids obtained in step (a).Step (b1) is adding the cross-linked sodium hyaluronate (HA) obtained instep (a6), to an about 0.7 to about 1.3% w/w aqueous sodium hydroxidesolution and mixing them together. Step (b2) is adding a cross-linkingagent to the resulting mixture from step (b1) and mixing together. Inthis step, about 10 to about 100 μl of the cross-linking agent may beadded per one gram of the cross-linked HA obtained in step (a). Step(b3) is a cross-linking reaction process of HA with the cross-linkingagent. This reaction may be performed in an about 40 to about 60

oven for about 8 to 10 hours. This cross-linking reaction in step (b3)may be performed for a time longer than step (a3) to induce generationof cross-linked hyaluronic acids in the form of particles. The obtainedcross-linked hyaluronic acids in the form of particles may be subjectedto dialysis in a buffer (step (b4)), followed by a neutralizationreaction with distilled water (step (b5)) and then precipitation ofhyaluronic acid as particles may be induced with about 95% ethanol (step(b6)) thereby obtaining cross-linked hyaluronic acids in powder form.

Step (c) is mixing the primary cross-linked product of sodiumhyaluronate in hydrogel form obtained in step (a) with the secondarycross-linked product of sodium hyaluronate in particle form obtained instep (b) in a variety of ratios, and preparing a final product viapulverization, filling, and sterilization. Step (c1) is mixing theprimary cross-linked product of sodium hyaluronate in hydrogel form andthe secondary cross-linked product of sodium hyaluronate in particleform in a ratio of about 9:1 to about 8:2 and dissolving the mixture ina buffer to prepare a combination of cross-linked hyaluronic acids. Step(c2) is pulverizing the combination of cross-linked hyaluronic acidsinto particles having a particle size of about 500 to about 750 μm,followed by filling a syringe with the combination of cross-linkedhyaluronic acids (step (c3)) and sterilizing the combination ofcross-linked hyaluronic acids-filled syringe (step (c4)) therebyobtaining a final combination of cross-linked hyaluronic acids having atarget viscoelasticity.

In some embodiments, the biocompatible material may be a synovial fluidsupplement including a combination of the cross-linked hyaluronic acidhaving an elasticity of 50 to 200 Pa and a viscosity of 20 to 100 Pa andthe cross-linked hyaluronic acid having an elasticity of 400 to 800 Paand a viscosity of 40 to 100 Pa, the combination in particle form havingan average particle size of about 500 to about 750 μm and having anelasticity of 100 to 150 Pa and a viscosity of 20 to 60 Pa.

In some embodiments, the biocompatible material may be a synovial fluidsupplement including a combination of the primary cross-linked productof hyaluronic acids having an elasticity of 50 to 200 Pa and a viscosityof 20 to 100 Pa and the secondary cross-linked product of hyaluronicacids having an elasticity of 400 to 800 Pa and a viscosity of 40 to 100Pa, the combination in particle form having an average particle size ofabout 500 to about 750 μm and an elasticity of 100 to 150 Pa and aviscosity of 20 to 60 Pa.

The synovial fluid supplement may have a viscoelasticity similar to thatof the human's real synovial fluid, and may be effectively used as asynovial fluid supplement. The synovial fluid supplement may includeboth the primary cross-linked product in hydrogel form and the secondarycross-linked product in particle form to thus provide lubricating andseparating functions for joints, and may serve a very similar functionlike the human synovial fluid. Due to the inclusion of both the primarycross-linked product in hydrogel form and the secondary cross-linkedproduct in particle form, the biocompatible material may be structurallystable, not easily decomposable in the human body, and have an effect asa synovial fluid supplement lasting for about 6 months or longer withone injection.

Another aspect of the present disclosure provides a method of preparinga combination of cross-linked hyaluronic acids according to any of theabove-described embodiments, the method including combining thecross-linked hyaluronic acid having an elasticity of about 50 to about200 Pa and a viscosity of about 20 to about 100 Pa and the cross-linkedhyaluronic acid having an elasticity of about 400 to about 800 Pa and aviscosity of about 40 to about 100 Pa, wherein the combining ratio isadjusted to meet a target viscoelasticity of the combination ofcross-linked hyaluronic acids to be prepared.

According to the method, a combination of cross-linked hyaluronic acidhaving a desired viscoelasticity may be obtained by adjustment of thecombining ratio of the cross-linked hyaluronic acids having anelasticity of about 50 to about 200 Pa and a viscosity of about 20 toabout 100 Pa and the cross-linked hyaluronic acid having an elasticityof about 400 to about 800 Pa and a viscosity of about 40 to about 100Pa. In some embodiments, by combining the cross-linked hyaluronic acidhaving an elasticity of about 50 to about 200 Pa and a viscosity ofabout 20 to about 100 Pa and the cross-linked hyaluronic acid having anelasticity of about 400 to about 800 Pa and a viscosity of about 40 toabout 100 Pa in a weight ratio of about 9:1 to about 8:2, a combinationof cross-linked hyaluronic acids having an elasticity of 100 to 150 Paand a viscosity of 20 to 60 Pa, which may lead to a similarviscoelasticity as the human synovial fluid, may be obtained. To preparea combination of cross-linked hyaluronic acids having a desiredviscoelasticity, the combining ratio of the primary cross-linked productof hyaluronic acids and the secondary cross-linked product of hyaluronicacids may be varied. Even when preparing a combination of cross-linkedhyaluronic acids having the same viscoelasticity, the combining ratio ofthe primary cross-linked product of hyaluronic acids and the secondarycross-linked product of hyaluronic acids may also be varied depending onthe molecular weight of hyaluronic acid source and the preparationconditions of the primary cross-linked product and the secondarycross-linked product (reaction temperature, reaction time, and thelike).

One or more embodiments of the present disclosure will now be describedin detail with reference to the following examples. However, theseexamples are only for illustrative purposes and are not intended tolimit the scope of the one or more embodiments of the presentdisclosure.

Preparation Example 1: Preparation of Cross-Linked HA in OrganicSolvent-Free Aqueous Alkaline Solution

After 10 g of sodium hyaluronate was mixed with a 1% w/w NaOH aqueoussolution, 50 μl of BDDE with respect to 1 g of sodium hyaluronate wasadded thereto and reacted at about 40° C. for about 3, 12, or 24 hours.

Preparation Example 2: Preparation of Cross-Linked HA inEthanol-Containing Aqueous Alkaline Solution (1)

After 10 g of sodium hyaluronate was mixed with a 1% w/w NaOH aqueoussolution containing 10% w/w of ethanol, 50 μl of BDDE with respect to 1g of sodium hyaluronate was added thereto and reacted at about 40° C.for about 3, 5, 12, or 24 hours.

Preparation Example 3: Preparation of Cross-Linked HA inEthanol-Containing Aqueous Alkaline Solution (2)

After 10 g of sodium hyaluronate was mixed with a 1% w/w NaOH aqueoussolution containing 10% w/w of ethanol, 50 μl of EGDGE with respect to 1g of sodium hyaluronate was added thereto and reacted at about 40° C.for about 24 hours.

Preparation Example 4: Preparation of Cross-Linked HA inEthanol-Containing Aqueous Alkaline Solution (3)

After 10 g of sodium hyaluronate was mixed with a 1% w/w NaOH aqueoussolution containing 10% w/w of ethanol, 50 μl of 1,6-hexanedioldiglycidyl ether with respect to 1 g of sodium hyaluronate was addedthereto and reacted at about 40° C. for about 24 hours.

Preparation Example 5: Preparation of Cross-Linked HA inMethanol-Containing Aqueous Alkaline Solution

After 10 g of sodium hyaluronate was mixed with a 1% w/w NaOH aqueoussolution containing 10% w/w of methanol, 50 μl of BDDE with respect to 1g of sodium hyaluronate was added thereto and reacted at about 40° C.for about 3, 12, or 24 hours.

Preparation Example 6: Preparation of Cross-Linked HA inIsopropanol-Containing Aqueous Alkaline Solution

After 10 g of sodium hyaluronate was mixed with a 1% w/w NaOH aqueoussolution containing 10% w/w of isopropanol, 50 μl of BDDE with respectto 1 g of sodium hyaluronate was added thereto and reacted at about 40°C. for about 3, 12, or 24 hours.

Experimental Example 1: Gelation Observation and ViscoelasticityMeasurement of Cross-Linked HA Prepared in Preparation Examples 1 to 6

It was identified whether gelation occurred or not in the cross-linkedHA of Preparation Examples 1 to 6 by naked-eye observation, andviscoelasticity measurements were performed. The viscoelasticitymeasurement was performed with a rotational rheometer (Kinexus ProRheometer, available from Malvern, Worchestershire, UK). Dynamicviscoelasticity was measured with a cone having a diameter of 20 mm at acone-plate distance (GAP) of about 0.5 mm. The temperature wasmaintained constant at about 25° C. until the end of the analysis. Theviscoelasticity measurement was performed with a control program in anoscillation mode at a frequency of about 0.1 to 5 Hz to measure astorage modulus G′ and a loss modulus G″. The storage modulus G′ meansenergy stored in a sample (elasticity behavior), and the loss modulus G″means lost energy (viscosity behavior). The results are shown in Tables1 and 2.

TABLE 1 Reaction condition Organic Reaction time at Reaction resultsolvent 40° C. Gelation Preparation None  3 hr No gelation occurredExample 1 12 hr Gelation occurred 24 hr Gelation occurred Preparationethanol  3 hr No gelation occurred Example 2  5 hr Gelation occurred 12hr Gelation occurred 24 hr Gelation occurred Preparation ethanol 24 hrGelation occurred Example 3 Preparation ethanol 24 hr Gelation occurredExample 4 Preparation methanol  3 hr No gelation occurred Example 5 12hr No gelation occurred 24 hr No gelation occurred Preparationisopropanol  3 hr No gelation occurred Example 6 12 hr No gelationoccurred 24 hr No gelation occurred

TABLE 2 Reaction condition Reaction result Organic Reaction timeViscoelasticity solvent at 40° C. Gelation G′ G″ Preparation None  3 hrNo gelation — — Example 1 12 hr Gelation 1789.7 300.8 occurred 24 hrGelation 2377.7 369.8 occurred Preparation ethanol  3 hr No gelation — —Example 2  5 hr Gelation 78.8 29.6 occurred 12 hr Gelation 379.2 63.3occurred 24 hr Gelation 1908.3 356.2 occurred

Referring to the results of Tables 1 and 2, it was found that gelationoccurred in the cross-linked hyaluronic acids of Preparation Example 1using the organic solvent-free aqueous alkaline solution and inPreparation Examples 2 to 4 using the ethanol-containing aqueousalkaline solution. However, the cross-linking reaction could not becontinued after the cross-linking reaction for 24 hours, due to browningof hyaluronic acids.

The cross-linked hyaluronic acids of Preparation Example 2 using theethanol-containing aqueous alkaline solution were found to have asignificantly low viscoelasticity, compared to the cross-linkedhyaluronic acids of Preparation Example 1 using the organic solvent-freeaqueous alkaline solution. This indicates that adding ethanol to anaqueous alkaline solution for a cross-linking reaction may suppress thecross-linking reaction, and consequentially result in cross-linkedhyaluronic acids having a reduced viscoelasticity.

In Preparation Examples 5 and 6 using an aqueous alkaline solutioncontaining a lower alcohol other than ethanol, even after thecross-linking reaction occurred for a long time, the cross-linkedhyaluronic acids in hydrogel form were not generated.

Example 1: Preparation of Primary Cross-Linked Product of HyaluronicAcids in Hydrogel Form

The preparation processes of Examples 1 to 3 were separately performedusing each of a high-molecular weight sodium hyaluronate (having amolecular weight of 2.5 Mda) and a low-molecular weight sodiumhyaluronate (having a molecular weight of 0.9 Mda). The high-molecularweight sodium hyaluronate (having a molecular weight of 2.5 Mda) and thelow-molecular weight sodium hyaluronate (having a molecular weight of0.9 Mda) both produced by fermentation with a Streptococcus spp.microorganism (Streptococcus zooepidemicus) were obtained from HanmiPharm Co., Ltd.

After sodium hyaluronate was added to a mixed solvent containing 1% w/wof a sodium hydroxide aqueous solution and 10% w/w of ethanol andcompletely dissolved, 50 μl of BDDE with respect to 1 g of sodiumhyaluronate was added thereto and mixed together. After the mixing, across-linking reaction was performed at a reaction temperature of about40° C. for about 5 hours. After termination of the reaction, theresulting primary cross-linked product of hyaluronic acids in hydrogelform were subjected to dialysis in a phosphate buffered saline (PBS)buffer, followed by washing with distilled water to remove BDDE. Theresulting neutralized hydrogel was subjected to extraction with a 95%ethanol aqueous solution thereby yielding a primary cross-linked productof hyaluronic acids in powder form.

Example 2: Preparation of Secondary Cross-Linked Product of HyaluronicAcids in Particle Form

The Primary Cross-linked product of hyaluronic acids obtained in Example1 was subjected to a secondary cross-linking reaction. The primarycross-linked product of hyaluronic acids in powder form obtained inExample 1 was mixed with a 1% w/w sodium hydroxide aqueous solution in aweight ratio of about 1:5 and then completely dissolved. 50 μl of BDDEwith respect to 1 g of the primary cross-linked product was added to theresulting reaction mixture and then mixed together. After the mixing, across-linking reaction was performed at a reaction temperature of about40

for about 8 hours. After termination of the reaction, the resultingsecondary cross-linked product of hyaluronic acids in particle form wassubjected to dialysis in a PBS buffer for about 12 to 24 hours, followedby washing with distilled water to remove BDDE. The resultingneutralized hyaluronic acids in particle form were subjected toextraction with a 95% ethanol aqueous solution to thereby yield asecondary cross-linked product of hyaluronic acids in powder form.

Example 3: Preparation of a Combination of Cross-Linked Hyaluronic Acids

The primary cross-linked product of hyaluronic acids in hydrogel formobtained in Example 1 and the secondary cross-linked product ofhyaluronic acids in particle form obtained in Example 2 were mixedtogether in a weight ratio of about 9:1 or about 8:2 in a PBS buffer toprepare a 2% w/w of mixed solution. The mixed solution was pulverized bypassing the mixed solution through a 500 μm-mesh sieve with physicalforce. The pulverized mixture was filled into a syringe, followed bysterilization at about 121° C. for about 20 minutes.

Experimental Example 2: Viscoelasticity Measurement of Combinations ofCross-Linked Hyaluronic Acids

The primary cross-linked product of hyaluronic acids in hydrogel formobtained in Example 1 and the secondary cross-linked product ofhyaluronic acids in particle form obtained in Example 2 were mixedtogether in a weight ratio of about 10:0, 9:1, 8:2, and 0:10 in a PBSbuffer to prepare 2% w/w of mixed solutions. The mixed solutions wereeach pulverized by passing them through a 500 μm-mesh sieve withphysical force. The viscoelasticity of each of the mixed solutions wasmeasured using a rotational rheometer (Kinexus Pro Rheometer, availablefrom Malvern, Worchestershire, UK). The results are shown in Table 3.

TABLE 3 Experiment condition Result Mixed ratio by weight ElasticityViscosity [Primary to (G′) (G″) Hyaluronic acids Secondary] [Pa/2.5 Hz][Pa/2.5 Hz] 2.5 MDa hyaluronic 10:0  78.8 29.6 acids 9:1 114.40 45.198:2 162.30 53.76  0:10 712.70 88.50 0.9 MDa hyaluronic 10:0  55.35 28.50acids 9:1 70.38 34.94 8:2 106.10 45.71  0:10 435.50 48.43

Referring to the results of Table 3, the combinations of cross-linkedhyaluronic acids had a different viscoelasticity depending on a mixedratio of the primary cross-linked product to the secondary cross-linkedproduct.

The combination of cross-linked hyaluronic acids obtained with theprimary cross-linked product and secondary cross-linked product from thehigh-molecular weight hyaluronic acid in a mixed ratio of about 9:1 byweight had an elasticity of about 114.40 Pa and a viscosity of about45.19 Pa. The combination of cross-linked hyaluronic acids obtained withthe primary cross-linked product and secondary cross-linked product fromthe low-molecular weight hyaluronic acids in a mixed ratio of about 8:2by weight had an elasticity of 106.1 Pa and a viscosity of 45.71 Pa.Accordingly, these two combinations of cross-linked hyaluronic acidswere found to have a similar viscoelasticity to the human synovialfluid.

The viscoelasticities of the cross-linked hyaluronic acid prepared underthe same conditions varied depending on the molecular weight of thestarting hyaluronic acid source. Even prepared under the samecross-linking conditions, the cross-linked hyaluronic acid from thestarting high-molecular weight hyaluronic acid source had a higherviscoelasticity than those from the starting low-molecular weighthyaluronic acid source. Therefore, to prepare a combination ofcross-linked hyaluronic acids having a desired viscoelasticity, thecombination ratio of the primary cross-linked product and the secondarycross-linked product may vary depending on the molecular weight of astarting hyaluronic acid source.

Experimental Example 3: Particle Size Analysis of a Combination ofCross-Linked Hyaluronic Acids

The particle size of the combination of cross-linked hyaluronic acidsobtained with the primary cross-linked product and secondarycross-linked product of the high-molecular weight hyaluronic acids in amixed ratio of about 9:1 by weight, found to have a similarviscoelasticity to the human synovial fluid in Experimental Example 2,was analyzed using a particle size analyzer (available from Microtrac,Montgomeryville, Pa.).

The resulting particle size distribution of the combination ofcross-linked hyaluronic acids is shown in FIG. 2.

Referring to FIG. 2, the combination of cross-linked hyaluronic acidswas found to have an average particle size of about 606 μm (D₁₀: 235.8μm, D₅₀: 553.5 μm, and D₉₀: 1009 μm).

Experimental Example 4: Cytotoxicity Assay

Cytotoxicity of the combination of cross-linked hyaluronic acidsobtained with the primary cross-linked product and secondarycross-linked product from the high-molecular weight hyaluronic acids ina mixed ratio of about 9:1 by weight, found to have a similarviscoelasticity to the human synovial fluid in Experimental Example 2,was evaluated by the MTT(3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium) assay.

According to the MTT assay, toxicity of an elution from the combinationof cross-linked hyaluronic acids for about 72 hours was evaluated,together with toxicity of elutions from Teflon as a positive controlgroup and Latex as a negative control group. The results of cellsurvival ratios obtained from the MTT assay ware shown in FIG. 3.

Referring to the results of FIG. 3, the cell survival ratios of Teflonand Latex were 100% and 37%, respectively. The combination ofcross-linked hyaluronic acids had a cell survival ratio of about 89.93%,which is above 80%, the lower limit of cytotoxicity. Therefore, thecombination of cross-linked hyaluronic acids as an embodiment of thepresent disclosure was found to have high biocompatibility.

While this invention has been particularly shown and described withreference to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the spirit and scope of theinvention as defined by the appended claims. The disclosed embodimentsshould be considered in descriptive sense only and not for purposes oflimitation. Therefore, the scope of the invention is defined not by thedetailed description of the invention but by the appended claims, andall differences within the scope will be construed as being included inthe present invention.

1. A method of preparing a cross-linked hyaluronic acid having anelasticity of about 50 to about 200 Pa and a viscosity of about 20 toabout 100 Pa, the method comprising cross-linking hyaluronic acid withan epoxy-based cross-linking agent having at least two epoxy functionalgroups in an ethanol-containing aqueous alkaline solution.
 2. A methodof preparing a cross-linked hyaluronic acid having an elasticity ofabout 400 to about 800 Pa and a viscosity of about 40 to about 100 Pa,the method comprising further cross-linking the cross-linked hyaluronicacid prepared using the method of claim 1 with an epoxy-basedcross-linking agent having at least two epoxy functional groups in anaqueous alkaline solution.
 3. The method of claim 1, wherein thehyaluronic acid is obtained by fermentation with a microorganism.
 4. Themethod of claim 1, wherein the hyaluronic acid includes hyaluronic acid,a salt of hyaluronic acid, or any mixtures thereof.
 5. The method ofclaim 4, wherein the salt of hyaluronic acid is selected from the groupconsisting of sodium hyaluronate, potassium hyaluronate, magnesiumhyaluronate, zinc hyaluronate, cobalt hyaluronate, tetrabutylammoniumhyaluronate, and any combinations thereof.
 6. The method of claim 1,wherein the hyaluronic acid comprises sodium hyaluronate having amolecular weight of about 100,000 to about 6,000,000.
 7. The method ofclaim 1, wherein the epoxy-based cross-linking agent having at least twoepoxy functional groups is selected from the group consisting of1,4-butanediol diglycidyl ether (BDDE), ethylene glycol diglycidyl ether(EGDGE), 1,6-hexanediol diglycidyl ether, propylene glycol diglycidylether, polypropylene glycol diglycidyl ether, polytetramethylene glycoldiglycidyl ether, neopentyl glycol diglycidyl ether, polyglycerolpolyglycidyl ether, digylcerol polyglycidyl ether, glycerol polyglycidylether, trimethylpropane polyglycidyl ether,1,2-(bis(2,3-epoxypropoxy)ethylene, pentaerythritol polyglycidyl ether,sorbitol polyglycidyl ether, and any combinations thereof.
 8. The methodof claim 1, wherein the ethanol-containing aqueous alkaline solutioncontains about 5 to about 13% w/w of ethanol.
 9. The method of claim 1,wherein the ethanol-containing aqueous alkaline solution is an aqueoussodium hydroxide solution of about 0.7 to 1.3% w/w containing about 5 toabout 13% w/w of ethanol.
 10. A combination of cross-linked hyaluronicacids comprising a cross-linked hyaluronic acid having an elasticity ofabout 50 to about 200 Pa and a viscosity of about 20 to about 100 Pa,and a cross-linked hyaluronic acid having an elasticity of about 400 toabout 800 Pa and a viscosity of about 40 to about 100 Pa.
 11. Acombination of cross-linked hyaluronic acids, the combination comprisinga cross-linked hyaluronic acid having an elasticity of about 50 to about200 Pa and a viscosity of about 20 to about 100 Pa prepared by a methodcomprising cross-linking hyaluronic acid with an epoxy-basedcross-linking agent having at least two epoxy functional groups in anethanol-containing aqueous alkaline solution; and a cross-linkedhyaluronic acid having an elasticity of about 400 to about 800 Pa and aviscosity of about 40 to about 100 Pa prepared by a method comprisingcross-linking the cross-linked hyaluronic acid having an elasticity ofabout 50 to about 200 Pa and a viscosity of about 20 to about 100 Pawith an epoxy-based cross-linking agent having at least two epoxyfunctional groups in an aqueous alkaline solution.
 12. The combinationof cross-linked hyaluronic acids of claim 11, the combination having anelasticity of about 100 to about 150 Pa and a viscosity of about 20 toabout 60 Pa.
 13. The combination of cross-linked hyaluronic acids ofclaim 12, wherein the cross-linked hyaluronic acid having an elasticityof about 50 to about 200 Pa and a viscosity of about 20 to about 100 Paand the cross-linked hyaluronic acid having an elasticity of about 400to about 800 Pa and a viscosity of about 40 to about 100 Pa are combinedin a weight ratio of about 8:2 to about 9:1.
 14. The combination ofcross-linked hyaluronic acids of claim 12, wherein the combination ofcross-linked hyaluronic acids is in the form of particles having anaverage particle size of about 500 to about 750 μm.
 15. A biocompatiblematerial comprising the combination of cross-linked hyaluronic acids ofclaim
 10. 16. The biocompatible material of claim 15, wherein thebiocompatible material is selected from the group consisting ofarthritis treatment implants, wrinkle fillers, cosmetic fillers, anddrug carriers. 17.-18. (canceled)